Fluorescent energy transfer is one mechanism which has been proposed for use in biosensor applications. In selecting optimum donor-acceptor dye pairs a number of the following criteria should be met: 1) low overlap between the absorption spectra of donor and acceptor so that the direct excitation of the acceptor by the laser line is minimal: 2) high overlap between the emission spectra of donor and absorption spectra of acceptor so that the energy transfer efficiency is maximal; 3) good separation between the emission maxima of the donor and acceptor so that the ratio of the two intensities can be taken; 4) the donor should be able to be excited with a laser; 5) the fluorescence maxima of both donor and acceptor should be at a wavelength higher than serum fluorescence; 6) both donor and acceptor should have high extinction coefficients and high fluorescence quantum yields to ensure maximum sensitivity.
In order to select optimum donor-acceptor pairs and properly characterize their fluorescence properties various aspects of the pairs and their interactions with proteins (e.g., antibodies and antigens) need to be considered including such issues as: 1) energy transfer properties in solution; 2) spectral separation to determine the energy transfer efficiency; 3) other interactions between dyes besides energy transfer; 4) better methods to determine the degree of labeling; 5) calculation of the characteristic distance for all the potential donor-acceptor pairs; 6) fluorescence lifetimes of individual donors and acceptors, as well as the donor-acceptor pairs.
In order to quantitate the degree of labeling of immunoglobulin G (IgG), for example, with fluorescent dyes, it is important to use an accurate analytical method.
Techniques generally used in the literature are based on the assumption that dyes have the same spectra properties before and after conjugation to the protein. For example, McKay et al. described two formula to calculate the degree of labeling of gamma globulin with fluorescein and rhodamine (I. C. McKay, D. Forman, and R. G. White, 1981 Immunology, 43, 591-602). They stated clearly that "no allowance has been made for any changes that may take place in the ratio of A.sub.280 /A.sub.495 on conjugation with protein". In determining the dye-to-protein ratio, Khanna calculated the protein content of the conjugates "from absorbance at 280 nm after subtracting the contribution due to free dye at this wavelength". (P. L. Khanna, 1988, in Nonisotopic Immunoassay, T. T. Ngo, eds., 211-229, plenum Press, New York). This procedure apparently assumes the spectra of conjugated dye is the same as the free dye. Recently, Wessendorf et al. developed a spectrophotometric method to determine the fluorophore-to-protein ratio in conjugates of 7-amino-4-methylcoumarin-3-acetic acid (AMCA) with mouse IgG. (M. W. Wessendorf et al. 1990, J. Histochemistry and Cytochemistry, 38, 87-94). They studied the effect of conjugation of the spectral properties of the dye by conjugating AMCA to the .epsilon.-amino group of N.sup..alpha. -acetyl lysine. The result was incorporated into the calculation procedure for the AMCA-to-protein ratio. This work is a significant improvement over the existing procedures. However, the model compound used in their study was a single amino acid, N.sup..alpha. -acetyl lysine, which is different from a protein. In addition, the conjugation of dyes to the .alpha.-amino groups was intentionally blocked by the use of acetylated lysine.
It has long been known that the .alpha.-amino terminus exhibits a lower pK.sub.a than the .epsilon.-amino side chain of lysine, and also that the pK.sub.a of the .alpha.-amino terminus tends to decrease with increasing length of the polypeptide chain. Thus, an oligopeptide is a much better model for both the .alpha.-amino and the .epsilon.-amino groups than an acetylated amino acid. Furthermore, Wessendorf et al. did not apply multiple linear regression to analyze their data. More importantly, the dye they used had an emission maxima at 350 nm which is of little practical use for biosensor applications that involve blood serum because of the severe interference from serum fluorescence at this wavelength.
The procedure described for this invention, however, has major advantages over the existing methods because it involves the use of a polypeptide as a model compound to mimic the protein-dye reaction. Moreover, the dyes studied in regard to the present invention are fluorescein and their derivatives which are widely used in biosensor applications.